The current invention identifies a new technology that may be usefull for eliciting potent cell-mediated and humoral immune responses to a candidate protein antigen for vaccine development.
Aluminum hydroxide generally elicits potent antibody responses to the candidate antigen and little if any cell-mediated immune responses.
Apostolopoulos et al. demonstrated the induction of strong cell-mediated immune responses to mucin 1 antigen (MUC 1) in mice when the animals were immunized with MUC1 antigen conjugated to oxidized mannan (ox-mannan). These studies are described in the following references: i) Production of anti-peptide specific antibody in mice following immunization with peptides conjugated to mannan. Okawa. Y, Howard. C. R. and Steward. M. W. 1992, J immunol. Methods 149: 127-131. Department of clinical sciences, London School of Hygiene and Tropical Medicine, London, UK; ii) Apostolopoulos, V., Pietersz. G. A., Loveland, B. E., Sandrin, M. S., and McKenzie, I. F 1995, Proc. Natl. Acad. Sci. USA, 92: 10128-10132. The Austin Research Institute, Studly Road, Heidelberg 3084, Victoria, Australia; and iii) Apostolopoulos, V., Loveland, B. E., Pietersz. G. A. and McKenzie. I. F. 1995, J Immunol. 155: 5089-5094. The Austin Research Institute, Studly Road, Heidelberg 3084, Victoria, Australia. However, the utility of ox-mannan as an adjuvant or immunomodulator in vaccine development to infectious agents has not been evaluated.
Antigens conjugated to oxidized or reduced mannan are likely to elicit strong cell-mediated and humoral immune responses to the candidate antigen. Alum has been used as an adjuvant that elicits good humoral immune responses and little if any cell-mediated immune responses.
It would be usefull to develop vaccine against human papillomavirus that may require an adjuvant capable of eliciting both humoral and cell-mediated immune responses to papillomavirus antigens. In this report, we describe the utility of ox-mannan in eliciting protective immune responses to infectious agents in cottontail rabbit papillomavirus model.
The human papilloma viruses (HPV) are nonenveloped, double-stranded DNA viruses, with over 75 types identified. Infection with HPV may result in development of genital condylomas and cervical neoplasia, and may be associated with as many as 90% of the cervical carcinomas. The papilloma viruses are species specific with respect to productive infection, and HPV infection in animals does not produce the disease. This necessitates the preliminary testing of candidate vaccines to be carried out in animal papillomavirus models. Cottontail rabbit papillomavirus (CRPV) was the first papillomavirus identified and also the first DNA virus associated with cancers. L1 is the major component of the virus capsid and expression of L1 in baculovirus or yeast results in the formation of virus like particles (VLPs). Immunization of animals with the major capsid protein (L1) VLPs results in the generation of neutralizing antibodies that recognize conformational epitopes formed when viral capsid proteins assemble into VLPs or virions. Although vaccination with VLPs alone is effective against challenge by infectious CRPV, it has no effect in containing pre-existing infection.
In this study we evaluated the utility of oxidized mannan as a carrier for vaccine development and immunotherapy, we conjugated oxidized mannan to CRPV early proteins (E-proteins) antigens expressed in E. coli and evaluated their efficacy in containing pre-existing infection.
Papillomavirus infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cows. Papillomaviruses infect epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection. Papillomaviruses are species specific infective agents; a human papillomavirus cannot infect a nonhuman animal.
Papillomaviruses may be classified into distinct groups based on the host that they infect. Human papillomaviruses (HPV) are further classified into more than 70 types based on DNA sequence homology (for a review, see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRC Press, Inc., 1990). Papillomavirus types appear to be type-specific immunogens in that a neutralizing immunity to infection to one type of papillomavirus does not confer immunity against another type of papillomavirus.
In humans, different HPV types cause distinct diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts in both normal and immunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25, 36 and 46-50 cause flat lesions in immunocompromised individuals. HPV types 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata of the genital or respiratory mucosa. HPV types 16 and 18 cause epithelial dysplasia of the genital mucosa and are associated with the majority of in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal. HPV6 and HPV11 are the causative agents for more than 90% of all condyloma (genital warts) and laryngeal papillomas. The most abundant subtype of HPV type 6 is HPV6a.
Immunological studies in animals have shown that the production of neutralizing antibodies to papillomavirus antigens prevents infection with the homologous virus. The development of effective papillomavirus vaccines has been slowed by difficulties associated with the cultivation of papillomaviruses in vitro. The development of an effective HPV vaccine has been particularly slowed by the absence of a suitable animal model.
Neutralization of papillomavirus by antibodies appears to be type-specific and dependent upon conformational epitopes on the surface of the virus.
Paprnomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode for up to eight early and two late genes. The open reading frames (ORFs) of the virus genomes are designated E1 to E7 and L1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 code for virus capsid proteins. The early (E) genes are associated with functions such as viral replication and cellular transformation.
The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. L2 protein is a minor capsid protein which has a predicted molecular weight of 55-60 kDa and an apparent molecular weight of 75-100 kDa as determined by polyacrylamide gel electrophoresis. Immunologic data suggest that most but not all of the L2 protein is internal to the L1 protein. The L2 proteins are highly conserved among different papillomaviruses, especially the 10 basic amino acids at the C-terminus. The L1 ORF is highly conserved among different papillomaviruses.
The L1 and L2 genes have been used to generate vaccines for the prevention of papillomavirus infections in animals. Zhou et al., (1991; 1992) cloned HPV type 16 L1 and L2 genes into a vaccinia virus vector and infected CV-1 mammalian cells with the recombinant vector to produce virus-like particles (VLP). Bacterially-derived recombinant bovine papillomavirus L1 and L2 have been generated. Neutralizing sera to the recombinant bacterial proteins cross-reacted with native virus at low levels, presumably due to differences in the conformations of the native and bacterially-derived proteins.
Recombinant baculoviruses expressing HPV6 L1, HPV11 L1, HPV16 L1, HPV18 L1, HPV31 L1 or HPV16 L2 ORFs have been used to infect insect SF9 cells and produce L1 and L2 proteins. Western blot analyses showed that the baculovirus-derived L1 and L2 proteins reacted with antibody to HPV16. The baculovirus derived L1 forms VLPs.
Carter et al., (1991) demonstrated the production of HPV 16 L1 and HPV16 L2 proteins by recombinant strains of Saccharomyces cerevisiae. Carter et al. also demonstrated the production of HPV6b L1 and L2 proteins. The HPV6b L1 protein was not full-length L1 protein. The recombinant proteins were produced as intracellular as well as secreted products. The recombinant L1 and L2 proteins were of molecular weights similar to the native proteins. When the proteins were expressed intracellularly, the majority of the protein was found to be insoluble when the cells were lysed in the absence of denaturing reagents. Although this insolubility may facilitate purification of the protein, it may hamper analysis of the native epitopes of the protein.
Recombinant proteins secreted from yeast were shown to contain yeast-derived carbohydrates. The presence of these N-linked oligosaccharides may mask native epitopes. In addition, the secreted recombinant proteins may contain other modifications, such as retention of the secretory leader sequence.
It would be useful to develop methods of producing large quantities of papillomavirus proteins of any species and type by cultivation of recombinant yeasts. It would also be useful to produce large quantities of papillomavirus proteins having the immunity-conferring properties of the native proteins, such as the conformation of the native protein.
The present invention is directed to recombinant papillomavirus proteins having the immunity conferring properties of the native papillomavirus proteins as well as methods for their production and use. The present invention is directed to the production of a prophylactic and therapeutic vaccine for papillomavirus infection. The recombinant late proteins of the present invention are capable of forming virus-like particles. These VLP are immunogenic and prevent formation of warts in an animal model. In addition, recombinant E-proteins are produced in E. coli and these proteins are presented so as to elicit a cell-mediated immune response. The present invention uses the cottontail rabbit papillomavirus (CRPV) and HPV type 6 (subtype 6a) as model systems.